Bottom Line:
GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10.Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo.This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

ABSTRACTGD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

fig2: GD3-loaded APCs induced GD3-reactive lymphocytes producing IL-4. Mice were injected with 105 GD3-loaded APCs or unloaded APCs into the footpad, as indicated. Popliteal and inguinal LN (a) or splenocytes (b) were collected on day 7 and tested for reactivity against irradiated syngeneic APCs loaded with GD3 or unloaded APCs (as negative control) as indicated. IL-4 production was detected by ELISPOT assay. Each point represents the mean of triplicate wells from an individual mouse; horizontal lines indicate mean values of five mice.

Mentions:
We wished to confirm that it was GD3 and not another molecule expressed by SK-MEL-28 that was responsible for immunization. To do this, mice were immunized by footpad injection with syngeneic APCs loaded with purified GD3; control mice were immunized with unloaded APCs. GD3-reactive lymphocytes were detected in the draining LN, such as popliteal and inguinal LN, (Fig. 2 a) as well as in the spleen (Fig. 2 b) of mice immunized with GD3-loaded APCs. GD3-reactive lymphocytes were not detected in control mice injected with unloaded APCs, or when splenocytes from immunized mice were tested against unloaded APCs in the ELISPOT assay. These studies confirmed that the immunizing molecule was GD3.

fig2: GD3-loaded APCs induced GD3-reactive lymphocytes producing IL-4. Mice were injected with 105 GD3-loaded APCs or unloaded APCs into the footpad, as indicated. Popliteal and inguinal LN (a) or splenocytes (b) were collected on day 7 and tested for reactivity against irradiated syngeneic APCs loaded with GD3 or unloaded APCs (as negative control) as indicated. IL-4 production was detected by ELISPOT assay. Each point represents the mean of triplicate wells from an individual mouse; horizontal lines indicate mean values of five mice.

Mentions:
We wished to confirm that it was GD3 and not another molecule expressed by SK-MEL-28 that was responsible for immunization. To do this, mice were immunized by footpad injection with syngeneic APCs loaded with purified GD3; control mice were immunized with unloaded APCs. GD3-reactive lymphocytes were detected in the draining LN, such as popliteal and inguinal LN, (Fig. 2 a) as well as in the spleen (Fig. 2 b) of mice immunized with GD3-loaded APCs. GD3-reactive lymphocytes were not detected in control mice injected with unloaded APCs, or when splenocytes from immunized mice were tested against unloaded APCs in the ELISPOT assay. These studies confirmed that the immunizing molecule was GD3.

Bottom Line:
GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10.Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo.This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

ABSTRACTGD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.